The liver plays a central role in the amino acid and carbohydrate metabolism of most vertebrate animals and hepatic gluconeogenesis represents an important bridge between the metabolism of these two classes of nutrients. Diabetes mellitus is a disease characterized by aberations in amino acid and carbohydrate metabolism, including severe hyperglycemia. The elevated levels of plasma glucose result from decreased utilization of glucose by extra-hepatic tissues and from increased glucose synthesis in the liver. The experiments described in the first half of this proposal are aimed at understanding the relationship between diabetes-induced hepatic amino acid transport and gluconeogenesis. The research will utilize the in vitro model system of rat hepatocytes, isolated by collagenase perfusion of the liver. The cells will be used to measure the rate of amino acid-dependent gluconeogenesis and the corresponding rate of transport for each amino acid, in the presence and absence of the other 19 naturally-occurring amino acids. Both the test substrates and the competing amino acids will be present in the assays at physiological concentrations. These experiments should provide new insight into the role of hepatic amino acid transport during development of diabetic-hyperglycemia. The experiments outlined in the second half of the proposal are designed to develop an assay system for the mRNA that codes for the glycoprotein responsible for the diabetes-induced System A transport activity. In the absence of specific tests for the System A carrier protein or its corresponding mRNA, poly(A)+ mRNA will be injected into the cytoplasm of Xenopus oocytes after which the cells will be used to monitor the expression of System A transport activity. The oocytes contain very little endogenous System A transport activity as measured by Na+-dependent MeAIB uptake. Antibodies and a cDNA probe for phosphoenolpyruvate carboxykinase, a gluconeogenic enzyme whose regulation is similar to System A, will be used to monitor the expression and stability in the oocyte of this hormone-regulated mRNA. These studies will provide a mechanism by which one can demonstrate, independent of inhibitors of macromolecular synthesis, transcriptional control of the System A gene. Development of the oocyte mRNA expression system for transport carriers such as System A will provide the opportunity to gain insight into the mechanisms and metabolic impact of transport regulation.